Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme

Background Abscisic acid (ABA) has been reported in controlling plant growth and development, and particularly dominates a role in resistance to abiotic stress. The Pyrabactin Resistance1/PYR1-Like /Regulatory Components of ABA receptors (PYR1/PYL/RCAR) gene family, of which the PYL9 is a positive regulator related to stress response in ABA signaling transduction. Although the family has been identified in grape, detailed VaPYL9 function in cold stress remains unknown. Results In order to explore the cold tolerance mechanism in grape, VaPYL9 was cloned from Vitis amurensis. The subcellular localization showed that VaPYL9 was mainly expressed in the plasma membrane. Yeast two-hybrid (Y2H) showed VaPCMT might be a potential interaction protein of VaPYL9. Through the overexpression of VaPYL9 in tomatoes, results indicated transgenic plants had higher antioxidant enzyme activities and proline content, lower malondialdehyde (MDA) and H2O2 content, and improving the ability to scavenge reactive oxygen species than wild-type (WT). Additionally, ABA content and the ratio of ABA/IAA kept a higher level than WT. Quantitative real-time PCR (qRT-PCR) showed that VaPYL9, SlNCED3, SlABI5, and antioxidant enzyme genes (POD, SOD, CAT) were up-regulated in transgenic tomatoes. Transcriptome sequencing (RNA-seq) found that VaPYL9 overexpression caused the upregulation of key genes PYR/PYL, PYL4, MAPK17/18, and WRKY in transgenic tomatoes under cold stress. Conclusion Overexpression VaPYL9 enhances cold resistance of transgenic tomatoes mediated by improving antioxidant enzymes activity, reducing membrane damages, and regulating key genes in plant hormones signaling and antioxidant enzymes. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03704-8.

A full understanding of the molecular mechanisms of cold tolerance will be conducive to plant breeding of cold tolerance species and provide cold tolerance candidate genes for the following researches.
ABA is an important phytohormone that regulates plant growth and development including cell elongation, seed dormancy, leaf senescence, fruit abscission, and stomatal closure [7,8]. In addition, ABA is also an important stress response phytohormone, and it's called as a 'stress hormone' . Many researches have verified that ABA functions as an endogenous messenger in abiotic stress responses, especially in drought, salinity and cold stresses, and plants keep a high ABA accumulation [9][10][11][12][13]. The PYR/PYL/RCAR proteins are currently considered as the typical ABA receptors according to structural, biochemical, and genetic evidences implemented by two independent research groups [10,11,14,15]. The PYR/PYL/RCAR proteins belong to Bet v 1-like superfamily members and include special STAR-RELATED LIPID-TRANSFER (START) related to metabolism, and hydrophobic compounds such as lipids, hormones, and antibiotics [16,17]. A number of studies found that PYR/ PYL/RCAR which was bound to ABA negatively regulated type 2C protein phosphates (PP2C) in ABA signaling transduction, in turn which negatively regulated SNF1-related protein kinase 2 (SnRK2). These three core components formed a double negative regulatory system in ABA signal transduction. Under normal growth conditions, PP2C inactivated SnRK2 through dephosphorylation, and ABA signal was non-activation. When plants were subjected to adverse environmental conditions, ABA production maintained a high level, ABA bound to PYR/PYL/RCAR proteins and promoted interaction function with PP2C to inhibit PP2C phosphatase activity, relieving the inhibition of PP2C on the protein kinase SnRK2, and SnRK2 was activated, and then phosphorylated downstream transcription factors or membrane proteins to arouse ABA signaling reaction [18,19].
There existed 14 PYL gene members in Arabidopsis thaliana, except PYL13, other gene sequences and structures were highly conserved, and could bind to ABA inhibiting PP2C enzyme activity [11,20]. Nine of the 14 PYR/PYL/RCAR family members were the most robustly co-purified proteins with ABI1. The data supported that ABI1 interacted with multiple members of the PYR/ PYL/RCAR family in Arabidopsis [21]. The knockout of AtPYL8 reduced the sensitivity of ABA on primary root growth and lateral root formation and the overexpression AtPYL9 promoted the lateral root elongation in the presence of ABA. AtPYL9 was found to interact with MYB77 and MYB44 in vivo to regulate auxin-responsive genes affecting roots growth [22,23]. Additionally, overexpression SlPYL9 accelerated tomato fruits ripening [24]. PYR/ PYL receptors regulated stomatal aperture highlights the relevance of the PYR/PYL pathway to cope with drought stress [14]. PYR/PYL/RCAR ABA receptors were close to abiotic stresses, RCAR12 and RCAR13 were confirmed to play positive roles in regulating extreme temperature, including cold and high temperature in Arabidopsis [25]. ABA receptors, RCAR11, RCAR12, RCAR13 and RCAR14 showed a hypersensitivity for ABA, increased genes expression related stress responses, as well as enhanced drought tolerance [26]. Under drought and low temperature treatments, the ectopic expression of OsPYL3 could enhance the cold and drought resistance of Arabidopsis thaliana, and OsPYL10 overexpression had the potential ability to improve both drought and cold stress tolerance of rice [27,28]. It has been reported that RCAR5/PYL11 was involved in response to cold stress by delaying seed germination and controlling stomatal closure via ABA-dependent and ABA-independent pathways, respectively [29]. Constitutive expression of PtPYRL1 and PtPYRL5 significantly enhanced the resistance to drought, osmotic, and cold stresses by positively regulating ABA signaling in Populus L. [30]. Overexpression AtPYL5 promoted drought tolerance [31,32]. Exogenous hormones induced AtPYL8 expression to improve drought stress [33]. MdPYL9 improved tolerance to drought stress in transgenic apple [34]. The expression level of GhPYL9-11A was higher in drought-tolerant cotton cultivars than in drought-sensitive cottons under drought treatment [35]. These functional achievements of PYR/PYL/RCAR showed that researches on stresses tolerance of PYR/PYL/RCAR mainly focused on drought. Although cold stress of PYL genes has been studied in several species, its knowledge was less than drought.
To date, a total of eight VvPYL genes were identified from the grape genome database, which could respond to cold, salt, and PEG stresses. The researcher successfully cloned six PYL genes from the wild Chinese V. yeshanensis ('Yanshan-1'), and overexpression VyPYL9 in Arabidopsis enhanced ABA sensitivity, and improved drought tolerance under ABA and drought treatments [36,37]. VvPYL1 modulated ABA signaling pathway by inhibiting PP2C expression [38]. VlPYL1 from 'Kyoho' promoted anthocyanin accumulation in grape berry skin [39]. There were most widely studies on CBF and COR genes related to cold stress in grape [40,41]. However, knowledge of PYLs related to cold stress is relatively narrow in grape. Therefore, in this study, VaPYL9 was cloned from Vitis Amurensis with strong cold tolerance, and it was ectopically expressed in tomatoes. Results showed that the VaPYL9 overexpression was induced by cold stress. The overexpression of VaPYL9 in tomatoes contributed to an increasing of the ability to scavenge reactive oxygen species and reduce membrane damage, and keep a high ABA level in transgenic tomatoes. Through RNAseq, the result showed VaPYL9 bound to ABA leading to a series of reactions in metabolism and biosynthesis, and mainly focused on MAPK and plant hormones signaling transduction paths under cold stress. In all, these findings indicate that VaPYL9 dominates a positive role in plant response to cold stress and may be an important candidate gene for molecular breeding for the improvement of stress tolerance in grape.

Detailed information and evolutionary analysis of VaPYL9
In order to have a basic understanding of VaPYL9 (GSVIVG01027078001), the physicochemical property was analyzed in supplementary Table S1. The online prediction found that isoelectric point (PI) was 6.38, the molecular weight was 20.05 KD, grand average of hydropathicity (GRAVY) was -0.193, instability index(II) was 37.65, and the aliphatic index(AI) was 95.67. The subcellular localization prediction showed that VaPYL9 was expressed mainly in cytoplasm. VaPYL9 protein was stable because instability index was less than 40. Protein was hydrophilic amino acid when GRAVY was negative value. Therefore, VaPYL9 protein had strong hydrophilicity. Phylogenetic tree analysis showed that PYL genes from grape, tomato, and Arabidopsis were divided into three subgroups, and VaPYL9 was clustered in III subgroup (Fig. 1). VaPYL9 and most tomato genes were clustered in a close evolutionary branch. Multiple sequences alignment showed that PYL genes had a polyketide cyclase/dehydrase and lipid transport domain Fig. 1 Evolution analysis of PYL genes from grape, Arabidopsis thaliana, and tomato. The phylogenetic tree was constructed using PYL sequences of grape, tomato, and A. thaliana in MEGA X, which was clustered into three subgroups. Different background colors represent different subgroups. VaPYL9 marked with red ellipse was in the III class. The green circle, blue circle, and pink circle represent grapes, A. thaliana, and tomato PYL genes, respectively (polyketide-cyc2, Pfam accession: PF10604) and highly conservative amino acid sequences ( Supplementary  Fig. S1). The finding indicated VaPYL9 might have a similar function to PYL genes of tomatoes.

Transient expression of VaPYL9 in tobacco
Based on the above online prediction, VaPYL9 was located mainly in the cytoplasm. To further verify the prediction result, the pART-CAM-EGFP-VaPYL9 vector was constructed. The pART-CAM-EGFP-VaPYL9 and pART-CAM-EGFP empty construct plasmids were transformed into Agrobacterium tumefaciens strain GV3101 ( Supplementary Fig. S2), and then were injected into tobacco leaves. The physical investigation found that VaPYL9 was not only located in plasma membrane and nucleus but also in cytoplasm (Fig. 2a).

The cloning of VaPYL9 and obtaining of transgenic tomatoes
To specifically investigate cold tolerance of the VaPYL9, the Open Reading Framework (ORF) with 591 amino acids was inserted into overexpression vector pCAM-BIA1301. The presence of VaPYL9 cDNA fragment was verified by PCR and sequenced in Escherichia coalition (DH5α) and Agrobacterium tumefaciens strain GV3101 ( Supplementary Fig. S3). Through a Agrobacterium tumefaciens medicated method, VaPYL9 fragment was successfully introduced into tomatoes and identified by PCR ( Supplementary Fig S4). Three stable and homozygous T3 lines (#2, #6, #9) were screened from transgenic tomato lines and named OE1, OE2, and OE3, respectively.

The interaction proteins screening of VaPYL9
To preliminary investigate interaction proteins of VaPYL9 from AD library-related cold stress, pGBKT7-VaPYL9  Fig. S5a). Self-activation and toxicity detection were detected in DDQ and DDQ (x-α-gal) plates. Bacterial plaque didn't become blue in DDQ and DDQ (x-α-gal) plates. This indicated that VaPYL9 had no self-activation and toxicity ( Supplementary Fig. S6a). Through the mating hybridization method, interaction proteins of VaPYL9 were preyed from AD library relatedcold stress. The presence of potential interaction proteins was verified by PCR using AD vector primers. Finally, VaPCMT (protein-L-isoaspartate O-methyltransferase 1) was screened by blast in NCBI. pGADT7-VaPCMT was commonly constructed and introduced into Y2H gold ( Supplementary Fig. S5b). Using the same procedure, self-activation and toxicity were detected, indicating that VaPCMT didn't produce self-activation and toxicity ( Supplementary Fig. S6b). pGBKT7-VaPYL9 and pGADT7-VaPCMT proteins were co-transformation into Y2H gold in QDO and QDO(x-α-gal) plates (Supplementary Fig. S7). Compared to negative and positive controls, bacterial plaque became gradually blue in DDQ and DDQ (x-α-gal) plates after 2 days. VaPCMT was a potential interaction protein of VaPYL9. Comprehensive above results, the findings showed that VaPYL9 and VaPCMT might be a potential interaction function in vivo (Fig. 2b).

Phenotype observation and degree of membrane damage in tomatoes under cold stress
To evaluate the role of VaPYL9 in response to cold stress, WT and OE lines were exposed to a long-term cold environment. qRT-PCR showed that relative expression of VaPYL9 was upregulated when grapes were induced by cold stress, and up to a maximum in 48 h (Fig. 3a). Compared with WT tomatoes, transgenic tomatoes were highly expressed in cold stress (Fig. 3b). Through phenotype observation in WT and OE lines under 4 ℃ cold stress for 0 h, 48 h, results indicated that WT and OE lines showed wilting difference but no significant in appearance, and WT showed cold damage and leaves coiled and wilted more heavily than OE lines (Fig. 3c). Some indexes involved in membrane damages were determined. In general, REL is frequently associated with abiotic stress, the membrane permeability tends to the increasing of REL when plants are exposed to abiotic stress. With the prolongation of a cold duration time, REL showed an increasing trend, indicating the degree of membrane damage was increasing in WT and OE lines. From 12 h, relative electrolyte leakage (REL) was quickly increasing. REL of OE plants was lower than WT under cold stress (Fig. 3d) showed an increasing trend in WT and OE with the time of cold stress increasing, but in all time points MDA of OE was lower than WT lines. Low-level MDA indicated that lipid peroxidation of membrane was relatively low (Fig. 3g). There were almost no differences in proline content between OE and WT under non-stress. Proline content of OE was higher than WT and reached a maximum at 48 h. High-level proline protected plant cells from cold stress (Fig. 3h). Taken together, the overexpression VaPYL9 improved the ability to scavenge reactive oxygen species (ROS), and reduce the degree of membrane lipid peroxidation and cell dehydration property to protect tomatoes from cold stress.

The changing of antioxidant enzymes activity and endogenous hormones level under cold stress
Catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) play an important role in maintaining the homeostasis of ROS metabolism and antioxidation defense system. The results showed that there was no significant difference in enzyme activities of OE and WT at 0 h, while CAT and POD activities of WT and OE lines showed an increasing trend in all cold time points. Interestingly, VaPYL9 expression tomatoes exhibited higher activities of SOD, CAT, and POD enzymes compared to WT under cold stress. These three enzyme activities reached a maximum under cold stress for 48 h (Fig. 4a). In all, the results indicated that VaPYL9 expression caused an increase of antioxidant enzymes activity, reducing ROS damage for tomatoes. Endogenous hormones were determined by High Performance Liquid Chromatography (HPLC). The content of ABA was increasing while IAA was decreasing with the prolongation of cold duration time under cold stress. VaPYL9 expression tomatoes exhibited higher content of ABA compared to WT under cold stress, and ABA content reached a maximum under cold stress for 48 h. On the contrary, VaPYL9 overexpression tomatoes exhibited lower content of IAA compared to WT under cold stress. ABA/IAA of OE was gradually increasing compared to WT with cold duration time increasing (Fig. 4b). The findings supported that VaPYL9 overexpression resulted in higher ABA content and ABA/IAA, promoting cold tolerance for plants.

qRT-PCR analysis
qRT-PCR was employed to perform expression level measurement under cold stress for 0 h, 48 h. The expressions of SOD (NM_001311084.1), CAT (NM_001247257), and POD (NM_001302921.2) key genes involved in antioxidant enzymes were significantly upregulated under cold stress. High expresssion levels of antioxidant enzymes led to the increase of SOD, CAT, and POD activities (Fig. 5a). Compared to WT, SlNCED3 (Solyc07g056570.1), a key rate-limiting enzyme gene in the process of ABA synthesizes, and SlABI5 (XM_010327616.3), a key gene in ABA signaling transduction was up-regulated in OE lines than WT under cold stress (Fig. 5b). Altogether, these findings indicated that VaPYL9 gene was highly expressed in transgenic plants when plants were subjected to cold stimulation, and VaPYL9 gene could positively respond to cold stress.

Transcriptome sequencing analysis
To investigate the role of VaPYL9 gene in regulating cold tolerance, the RNA-Seq analysis was performed using the six-week-old transgenic and WT tomato seedlings incubated at 4 ℃ for 48 h and identified differentially expressed genes (DEGs). Basic information of sequencing data was listed in Table 1, a total of 44 Gb clean data was gained, GC content of every sample was up to over 40%, and base percentage Q30 of every sample was over 94.43%. According to the statistics of the comparison results, the comparison efficiency between reads of each sample and reference genome ranged from 95.00% to 95.97%. A total of 1270 DEGs were found in WT vs OE lines subset based on the criteria fold change > 2 and P < 0.05, of which 740 DEGs (VaPYL9-activated genes) were up-regulated, 530 DEGs were down-regulated (VaPYL9-repressed genes) (Fig. 6a, b). GO (Gene Ontology) showed that key functional annotations were associated with transcription factor activity, oxidoreductase activity, signaling receptor activity, abscisic acid binding, peroxidase activity, and protein serine/threonine kinase activity (Fig. 6c). KEGG analysis found that VaPYL9 overexpression mediated MAPK and plant signaling transduction paths to regulate cold tolerance. There were 34 DEGs in MAPK path, of which 25 DEGs were upregulated in OE compared to WT (Fig. 6d). MAPK activated the upstream ABA signaling to function in cold defense, MAPK17/18 was highly expressed in adapting to stress (Fig. 6d). There were 43 DEGs in plant signaling transduction path, of which 29 DEGs were up-regulated in OE compared to WT (Fig. 6e). Key PYR/PYL and MYC2 genes were up-regulated in OE compared to WT. Transcription factors mainly were involved in WRKY, and were up-regulated in OE lines than WT (Fig. 6f ).
Through qRT-PCR verification, MAPK17/18, PYR/PYL, MYC2, WRKY, PYL4, PR5 and GsSRK, some key genes involved in MAPK, plant signaling transduction, signaling receptor activity, and transcription factors activity were up-regulated in transgenic under cold stress (Fig. 5c). In summary, these data demonstrated that VaPYL9 played a positive role in cold stress tolerance in tomatoes mediated by MAPK and plant signaling transduction paths, improving transcription factors and signaling receptor activity (Fig. 6h).

ABA regulates cold tolerance in plant
Low temperature is a serious threat to crop yield stability, plant growth, quality improvement, and plant geography distribution [42]. To adapt to cold stress, a variety of biochemical and physiological processes have formed in plant. Plants modulate multiple morphological and physiological processes to maintain cellular and metabolic homeostasis, including changes in phytohormone homeostasis [43]. ABA is an important stress hormone that adjusts plant stress such as extreme temperature, salt, drought [1]. ABA has been demonstrated to be involved in the cold stress response through the regulation of a series of specific stress-responsive genes, and ABA can coordinate several phytohormones that integrated with cold signaling to regulate cold stress [13]. In recent years, an increasing number of studies have attached the importance of studying the mechanisms of ABA in cold tolerance. Exogenous ABA can improve the content of proline and soluble sugar, enhance water retention, reduce membrane lipid peroxidation, protect membrane integrity, and reduce photosynthetic characteristics [44,45]. In addition, exogenous ABA application altered the expression of or related-cold and ABA genes, improving resistance to cold in Bermudagrass, maize, and cucumber [46][47][48]. Here, through previous RNA-seq, ABA receptor VaPYL9 was selected from DEGs related to cold stress to further verify the function of cold tolerance.

The ABA receptors PYLs were involved in cold stress
The function of PYL proteins on ABA-mediated stress responses has been well studied [18]. ABA receptors OsPYL3, OsPYL10, and AtPYL11 enhanced the cold tolerance of plants [27][28][29]. Vitis Amurensis Rupr, which has strong cold hardiness can withstand -35 ℃ [49]. In this study, VaPYL9 gene from Vitis Amurensis Rupr was cloned, evolutionary analysis showed that PYLs could be classified into three subfamilies (Fig. 1). The result was consistent with clusters in Brassica napusin and rice [50,51]. The alignment of amino acid sequences revealed that these PYLs were highly conserved (Fig. 1b), especially amino acid residues that involved in ABA binding and PP2C interaction [52]. VaPYL9 had a polyketide-cyc2 domain related to polyketide cyclase/dehydrase and lipid transport as the same result in the previous study [53]. Most AtPYLs were mainly localized to the cytoplasm and the nucleus, and transduced ABA signals to activate a series of responses to various environmental factors [10,11]. The same location result was found in this study, through predication online and physical transient transformation in tobacco, which showed that the VaPYL9 was also localized in the cytoplasm and nucleus (Fig. 2a). When plants were subjected to abiotic stress, ABA was synthesized and bound to PYL proteins, and the ABA-PYL complex interacts with PP2Cs to initiate ABA signal transduction [31,54]. It has been reported that PYLs interacted with specific PP2C members in an ABA-dependent or ABA-independent manner [20]. In the present study, through screening of the Y2H method, VaPCMT was identified to be a potential protein with VaPYL9 commonly to mediate cold tolerance (Fig. 2b), and PCMT was related to oxidative stress in the previous study [55].

Overexpression of VaPYL9 improved cold tolerance mediated by scavenging reactive oxygen species, reducing membrane damage in tomato
Many previous evidences have shown that PYLs play crucial roles in ABA-mediated responses to cold stress [27][28][29]. Previous research reported that AtPYL9, the homolog sequence of VaPYL9 in Arabidopsis, enhanced ABA sensitivity and drought stress when expressed to high levels in transgenic Arabidopsis [56]. But PYL9 function was seldom involved in cold stress compared to drought. To preliminarily explore the function of VaPYL9 in response to cold in grape, overexpressing-VaPYL9 tomatoes lines were generated to evaluate the response of these transgenic plants under cold stress.
To have a good understanding of mechanisms associated with VaPYL9-induced cold tolerance, changes in several physiological and biochemical characteristics which are known to be associated with response to cold stress were determined. When plants are exposed to abiotic stress leading to an increasing of ROS production, which can cause oxidative damage to cellular components, and the intracellular ROS level is considered as an important indicator of cell death in response to stress conditions [57]. Here, results showed that REL, H 2 O 2 and MDA accumulation in WT plants was higher and proline was lower than in the transgenic lines after cold stress (Fig. 3d, e, f, g, h), suggesting that VaPYL9 expression is related to oxidative stress responses. To protect cells and membrane integrity from effects of excessive ROS, plants have evolved a series of complex antioxidant defense mechanisms to keep the homeostasis of ROS metabolism, such as SOD, CAT, and POD [58][59][60]. The results of the present study showed that VaPYL9 overexpression, transgenic tomatoes exhibited higher activities of SOD, CAT, and POD enzymes compared to WT under cold stress (Fig. 4a), suggesting that the reducing ROS in transgenic tomatoes may result from increasing ROS metabolism by these enzymes. qRT-PCR showed SOD, CAT and POD gene of OE lines was up-regulated under cold stress compared to WT (Fig. 5a). Coincidentally with plant stress, overexpression of GhPYL9-11A, PtPYL1, and PtPYL5 genes also caused an increasing in activities of antioxidant enzymes and the ability to scavenge ROS [30,35,61]. SlNCED3, a key rate-limiting enzyme gene in the process of ABA synthesizes, and SlABI5, a key gene in ABA signaling transduction was up-regulated in OE lines under cold stress compared to WT (Fig. 5b). Consistence with the result that NCED3 promoted ABA biosynthesis under stress and ABI5 was related to multiply hormones adapting to various stresses [62,63]. With the prolongation of a cold duration time, the ratio of ABA/ IAA was increased in OE lines than in WT (Fig. 4b). The result was similar to the previous study, the ratio of ABA/ IAA was increased in sugarcane leaves under cold stress [64]. The result showed that the increasing activities of antioxidant enzymes, reducing membrane damage, improving the ability to scavenge ROS, and high ABA content constitute an important physiological mechanism in plants driven by VaPYL9 overexpression under cold stress.

VaPYL9 improved cold tolerance mediated by MAPK pathway and multiply hormones coordination
Additionally, RNA-seq found a total of 1270 DEGs were found in WT vs OE lines subset, including 740 DEGs (VaPYL9-activated genes) that were up-regulated, and 530 DEGs were down-regulated (VaPYL9-repressed genes) (Fig. 6a, b). Through the KEGG map, the result showed overexpression VaPYL9 activated MAPK pathway and ethylene, and ABA commonly promoting adaption to cold stress in plant (Fig. 6d, e). MAPK was induced by ABA during abiotic stress. A combination of MAP3Ks, MAP3K17/18, MAP2K MKK3 and MAPKs MPK1/2/7/14 MAPK cascade related to PYR/PYL/RCAR mediated ABA signaling pathway, MPK1 and MPK2 may also phosphorize many ABA effector proteins [65,66]. Similarly, PYR/PYL and MAP3K17/18 were up-regulated in transgenic plants under cold stress (Fig. 6d). PYL6 responded to JA by interacting with MYC2. In the presence of ABA, the interaction of the two become a strength between ABA and JA signaling pathways [67]. The ERF1 binding to response element to regulate abiotic stress and mediated ROS signaling to response salt stress [68,69]. Coincidentally, in this study MYC2 indirectly regulated ERF1 to connect JA and ethylene enhancing defending stress, the two were up-regulated in overexpression VaPYL9 induced by cold stress (Fig. 6d, e). In all, VaPYL9 response to cold stress probably thereby MAPK and hormone signaling paths.

Conclusion
In summary, a working model of the role of VaPYL9 in the response of tomatoes to cold stress was proposed (Fig. 7). VaPYL9 overexpression induces the high expression of SlNCED3 and SlABI5 leading to ABA accumulation. VaPYL9 might be active in MAPK path of the downstream of ABA signaling response RNA-seq indicated that MAPK and other hormones also might be involved in improving cold response. Our works suggest that the VaPYL9 may be a good candidate gene for breeding cold-tolerant crops.

Plant materials and growth conditions
One-year-old grapevines (Vitis amurensis Rupr. var. 'Zuoshan 1') derived from the vineyard located in Gansu Agricultural University (Lanzhou, Gansu, China), were used to extract RNA. Tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum cv. 'Micro-Tom') seeds were purchased from Nanjing Fengshuo Horticulture Ltd., Company (Nanjing, Jiangsu, China). Tomato seeds were soaked using sterilized distilled water for 3 h, surface sterilized in 75% (v/v) ethanol for 30 s, and then treated with 2% (v/v) NaClO for 10 min, followed by four washes with sterilized distilled water. Seeds were planted on 1/2 Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose and 6.5 g/L agar, pH was 5.8-6.0. Tomatoes with two cotyledons expansion about six days were used for genetic transformation. Transgenic tomato seeds (Transgenic generation 3, T3) and WT seeds were soaked in a conical flask with warm water for 30 min, and then placed on a shaking table until most seeds showed white sprouts. Seeds with white sprouts and tobacco seeds were planted in plastic plots with seedling substrate containing a mixture of soil and vermiculite (3:1), and cultured in growth chambers at 28 °C, 8 h, the light at 135 μmol·m −2 ·s −1 /20 °C, 16 h, dark growth condition. The six-week-old T3 transgenic and WT tomato seedlings were used for the following cold assay. Fourweeks old tobacco seedlings were used for transient transformation.

Transient expression in tobacco
Four-weeks-old tobacco leaves were used to perform transient transformation for observing VaPYL9 expression location in plant cell. The first strand cDNA of grape phloem was employed as a template to clone VaPYL9 gene. The purified cDNA fragment was combined with pART-CAM-EGFP vector to form a fusion protein vector. The fusion plasmid with pART-CAM-VaPYL9-EGFP was introduced into Agrobacterium tumefaciens strain GV3101. pART-CAM-VaPYL9-EGFP Agrobacterium tumefaciens liquid was injected into tobacco leaves back, and then was incubated in the dark at 26 °C for 12 h. After dark incubation, injected tobaccos were placed in the light at 26 °C for 24 h. At last, green fluorescence was motivated in tobaccos and tobaccos leaves were snapped by confocal microscopy using laser scanning confocal microscope (Olympus FV1000 viewer) [72].

Y2H protein binding assay
The CDS sequence of VaPYL9 was cloned into pGBKT7 and fused to the DNA-binding domain to generate a bait construct. Primer sequences used in these assays are listed in Supplementary Table S1. The recombinant plasmid was transformed into Y2H Gold yeast strain, which was cultured on double dropout (DDO) medium (SD/ − Leu/ − Trp) for 3 days at 30 ℃ to detect self-activation and toxicity. PCR was used to verify the presence of VaPYL9 in the Y2H Gold strain. The Mating hybridization method was adopted to screen potential interaction proteins from the AD library related to cold treatment which was constructed and preserved in Physiology and Biotechnology Laboratory (Gansu Agricultural University, China). Screened potential protein sequences were cloned into pGADT7 and fused to activation domain sequences to generate prey construct. Self-activation and toxicity detection were detected using the same method. The two constructs recombinant plasmids were co-transformed into Y2H Gold strain yeast cells and then cultured on a double dropout (DDO) medium (SD/ − Leu/ − Trp) at 30 ℃ for 3 days. After Co-transformation, the colonies were transferred to a quadruple dropout (QDO) medium (SD/ − Leu/ − Trp/ − Ade/ − His) with 10 mM 5-Bromo-4-chloro-3-indolyl-α-D-pyrangalactoside (X-α-Gal) to examine potential physical binding and interaction. Combinations of pGADT7-T with pGBKT7-53 and pGBKT7-Lam were served as positive and negative controls, respectively. All plates were incubated at 30 °C for 4 days and then photographed.

Cloning of VaPYL9 and obtaining of transgenic tomatoes
Based on our project's previous transcriptome sequencing on Amur grape phloem under cold stress, VaPYL9 was screened and identified as a key gene from grape in cold stress. To further verify VaPYL9 gene function, the full-length VaPYL9 cDNA was cloned using grape phloem cDNA as a template into the overexpression vector pCAMBIA1301 digested by NcoI and BstEII enzymes, and Escherichia coalition (DH5α) carried recombinant plasmid was sequenced by Sangon Biotech (Shanghai, China) Co. Ltd. After successfully sequenced, the recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101. Agrobacterium tumefaciens strain GV3101 with overexpression pCAMBIA1301-VaPYL9 were transformed into WT tomato plants by the Agrobacteriummediated leaf disk transformation method as described previously [73]. Gained sterile tomato cotyledons were cut as infection materials. Cut cotyledons were planted in MS (30 g/L sucrose + 6.5 g/L + 1 mg/L KT) to preincubate in dark condition for 2 days. Prepared infection bacteria solution using 1/2 MS suspension was used to immerse preincubated cotyledons for 5 min. Then infected cotyledons were coincubated for 3 days in dark condition. After cocultivation, they were transferred to generate callus and adventitious buds in MS (30 g/L sucrose + 6.5 g/L agar + 0.2 mg/L IAA + 2 mg/L ZT + 350 mg/L Timentin + 30 g/L hygromycin (Hyg). When the length of adventitious buds was about to 3 cm, cutting adventitious buds were transferred into MS (30 g/L sucrose + 6.5 g/L agar + 0.05 mg/L IAA + 350 mg/L Timentin + 30 g/L Hyg) to form roots. Transgenic plant lines were screened for 30 mg/L Hyg resistance and identified by the special primer according to instruction of FTL MIX (MF848) kit (Mei5 Biotechnology, Beijing, China Co., Ltd). Homozygous transgenic plants were selected in T1 generation derived from T0, and were stably maintained up to T3 generations. Ultimately, homozygous and stable T3 lines were gained.

Cold assay
Three stable T3 transgenic lines were selected from previously gained tomatoes. By cooling plant culture chamber up to 4 °C step by step, some growth well and consistent six-week-old tomato plants including transgenic plants and WT plants were selected and placed in the culture chamber keeping 4 °C for 48 h. Tomato and grape leaves were collected in 0 h, 6 h, 12 h, 24 h, 48 h, quickly frozen in liquid nitrogen and stored at − 80 °C for qRT-PCR and determination of physiological indexes related to cold tolerance. Three biological replicates were applied in this experiment.
Determination of H 2 O 2 , MDA, proline contents and antioxidant enzyme activities H 2 O 2 and malondialdehyde (MDA) content and catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities were performed as described in the instruction of the commercial ELISA kit purchased from Suzhou Comin Biotechnology Co.ltd (Suzhou, Jiangsu, China) and reaction mixtures were measured in Multimode Reader (Spark ® Multifunctional microplate detector, TECAN, Pudong, Shanghai). The proline contents were determined as previously described [74].

Determination of relative electrolyte leakage and content of IAA and ABA
The relative electrolyte leakage (REL) was measured using a model DDS307A device (Shanghai Leica Instrument) and the ratio of C1 (value of Leaf before boiling) to C2 (value of Leaf after boiling) was calculated as previously described [75]. 20 round leaves were gained using punches, and placed in glass test tubes with 15 mL ultrapure water. After 12 h, C1 was detected by a conductivity instrument. Then boiling for 15 min, C1 was detected. C1/C2% was viewed as relative electrical conductivity value. The content of IAA and ABA was determined by High Performance Liquid Chromatography (HPLC) mentioned in previous experiments [76]. Transgenic and WT Tomatoes leaves were ground and extracted using 5 mL 80% (V/V) chromatographic methanol at 4 ℃ for 12 h. During extraction, samples were constantly shaken to make hormone sufficiently dissolve. After centrifuged at 8000 rpm, 4 ℃,10 min, supernatants were transferred in new tubes. Repeat steps above once, supernatants were transferred in the same tube and constant volume was up to 10 mL. 2 mL supernatants were evaporated to dryness using a rotary evaporator. After evaporated to dryness, residue samples were thawed using 2 mL 50% (V/V) chromatographic methanol. HPLC condition was listed: Symmetry C18 chromatographic column (4.6 mm* 250 mm、5 μm), flowing phase (10% chromatographic methanol and 90%, 0.1% phosphoric acid), flow velocity (1 mL/min), sample volume (10 μL), determination waves length (254 nm), the temperature column (30 ℃). Hormones were detected in American Waters Acquity Arc high performance liquid chromatography.
The DAB and NBT staining 3, 3, 9-Diaminobenzidine (DAB) and Nitrotetrazolium Blue Chloride (NBT) staining methods were performed as previously described [76,77]. 10 mM 2-(N-Morpholino) ethanesulfonic acid (MES) was dissolved in distilled water and PH was 5.5. 1 mg/L DAB (Sigma-Aldrich) was dissolved in MES Buffer Solution and PH was 5.5. 0.5 mg/L NBT (Sigma-Aldrich) was dissolved in distilled water and PH was 5.8. Prepared DAB and NBT stain solution were stored at 4 ℃ and kept in a dark place. The destaining solution was ethanol: lactic acid: glycerin (3:1:1). Detached Leaves of tomatoes were immersed in culture dishes containing DAB and NBT staining solution for 12 h. The stained leaves were boiled with 95% ethanol for 10 min and leaves were decolorized with a destaining solution for 2 h. Decolorized leaves were placed in culture dishes with distilled water and photoed in EPSON Scan of root scanner (Regent company, Canada).

RNA extraction and qRT-PCR
Total RNA was extracted from the leaves of grape and transgenic and WT tomato plants through RNAprep Pure Plant Kit (TIANGEN, Beijing, China). RNA was reversed as cDNA using the Prime Reverse Transcriptase Kit (Takara Bio, Shiga, Japan). Reversed cDNA was stored at − 20 °C and used as templates to amplify genes and perform qRT-PCR. All primers were designed on an online website and synthesized by Sangon Biotech (Shanghai) Co. Ltd (Supplementary Table S2). qRT-PCR was performed through the Light Cycler ® 96 real-time PCR system (Roche, Switzerland). The VvNAPDH (Gen-Bank accession no.CB973647) and SlActin (GenBank accession U60480) gene were used as internal controls. The 20 µL reaction solution contained 2 µL cDNA, 2 µL primers, 10 µL SYBR Green Master Mix Reagent (Takara Bio, Shiga, Japan), and 6 µL ddH 2 O. PCR amplification conditions were set as 50 cycles of 95 °C for 30 s, 95 °C for 10 s, 58 °C for 30 s, 72 °C for 20 s. The relative expressions of the genes were obtained from three biological replicates and calculated using the 2 −△△Ct method [78].

RNA-seq library preparation and sequencing in tomatoes under cold stress
Based on comprehensive data analysis in tomatoes after cold stress, transgenic and WT tomato leaves at 4 ℃ for 48 h were screened for RNA-seq. RNA extraction was conducted as described above. RNA-seq was commissioned by Biomarker Technologies Co., Ltd. (Beijing, China). Three biological replicates of each sample were used for RNA-seq analysis. RNA quality and concentration were determined by agarose gel electrophoresis using NanoPhotometer spectrophotometer (Implant, Germany) and Agilent 2100 Bio-Analyzer (Agilent Technologies, USA). RNA samples with inspection qualified were used to construct the library. Eukaryotic mRNA was enriched by magnetic beads with Oligo (dT). The mRNA was randomly interrupted by Fragmentation Buffer. Using mRNA as a template, the first cDNA strand was synthesized using random hexamers, and then the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. The cDNA strand was purified using AMPure XP Beads. The purified double-stranded cDNA was then terminally-repaired, added Poly-A and linked to a sequenced adaptor. AMPure XP Beads were then made segment size selection. Finally, the cDNA library was obtained by PCR enrichment. The libraries were sequenced on an Illumina the Novaseq6000 platform. FPKM value of each gene was calculated to estimate gene expression levels [79]. Gene expression difference was estimated using the DESeq2 [80]. The differentially expressed genes (DEGs) were screened by setting false discovery rate (FDR) < 0.05 and |Log2 FC (fold change)|≥ 2 as thresholds screening measurement. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs was implemented by the cluster Profiler [81,82].

Statistical analysis
All data were determined in three independent biological replicates for each experiment. Data analyses were done by using one-way analysis of variance (ANOVA) in SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). Duncan's multiple range tests were employed to test significant differences (P < 0.01). Origin 9.0 was used to draw diagrams.